WO2021136256A1 - 一种得到高纯度异源抗体的方法 - Google Patents

一种得到高纯度异源抗体的方法 Download PDF

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WO2021136256A1
WO2021136256A1 PCT/CN2020/140756 CN2020140756W WO2021136256A1 WO 2021136256 A1 WO2021136256 A1 WO 2021136256A1 CN 2020140756 W CN2020140756 W CN 2020140756W WO 2021136256 A1 WO2021136256 A1 WO 2021136256A1
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antibody
heavy chain
heterologous
protein
heterologous antibody
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周易
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周易
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific

Definitions

  • the present invention belongs to the field of antibody engineering, and specifically relates to a method for obtaining a high-purity heterologous antibody, which contains two heavy chains with different amino acid sequences.
  • IgG type bispecific antibodies have similar structure, physicochemical properties and Fc segment functions to common antibodies.
  • an IgG type bispecific antibody consists of two heavy chains with different amino acid sequences (i.e. heavy chain against antigen A and heavy chain against antigen B) and two light chains with different amino acid sequences (i.e. light chain against antigen A and heavy chain against antigen B).
  • Anti-antigen B light chain) composition When 4 polypeptide chains are combined, there are 8 different combinations, of which only one is the desired target antibody molecule. However, it is extremely inefficient and difficult to separate and purify the target molecule from 8 kinds of molecules.
  • Document 1 reports a method using a common heavy chain and a combination of two different light chains.
  • the limitation of this method is that it is very difficult to screen and obtain the heavy chain of anti-antigen A and heavy chain of anti-antigen B with exactly the same amino acid sequence.
  • Documents 2 and 3 reported the use of a common light chain and a combination of two different heavy chains.
  • the limitation of this method is that there are 3 combinations of two different heavy chains, including one heterologous antibody (bispecific antibody) and two homologous antibodies (impurities). In theory, the ratio is usually only about 50%. If the efficiency of the heterologous association of the two heavy chains is increased, the expression efficiency of the target bispecific antibody can be increased to 90-95% (Document 4).
  • Literature 5 reported the following method: by introducing amino acid mutations in the variable regions of the two heavy chains to give the two homologous antibodies and the target bispecific antibody different isoelectric points, thereby passing Purified by ion exchange chromatography to obtain the target bispecific antibody.
  • ion exchange chromatography is not a necessary purification step. Adding ion exchange chromatography can increase the purity of the target antibody, but it also reduces the final yield, leading to the preparation of Increased costs.
  • Protein A affinity chromatography is a necessary purification step for IgG antibodies. How to obtain high-purity bispecific antibodies by protein A affinity chromatography has become a new research direction.
  • Literature 6 reported a method: using the heavy chain of mouse IgG2a (which can bind to protein A) and the heavy chain of rat IgG2b (not bound to protein A) to form a bispecific antibody, only purified by protein A Purify the target bispecific antibody to 95% purity.
  • the limitation of this method is that the heavy chain constant region of mice and rats is extremely immunogenic (Reference 7), and the antibody Catumaxomab (Catumaxomab) prepared by this method has a half-life of about 2.1 in humans.
  • Literature 9 reported another method for obtaining high-purity bispecific antibodies using protein A purification: introducing a mutation in one of the heavy chain constant regions that constitute the bispecific antibody. Specifically, mutating His at position 435 of EU numbering to Arg, thereby changing the binding ability to protein A, can purify the target antibody to a purity ranging from 93 to 99.6%.
  • Literature 1 Fischer, N., et al. (2015). Nat Commun 6: 6113.
  • the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for obtaining high-purity heterologous antibodies by only using protein A affinity chromatography.
  • the present invention by introducing new mutations in the constant region of the heavy chain, changing the binding force with protein A, and then by protein A affinity chromatography, bispecific antibodies with a purity of >99% can be obtained.
  • the first aspect of the present invention is to provide a method for obtaining a high-purity heterologous antibody.
  • the heterologous antibody contains two heavy chains with different amino acid sequences.
  • a affinity chromatography purification can obtain high-purity bispecific antibodies, wherein the positions of the above-mentioned amino acids are determined according to the EU index of the KABAT number.
  • the positions of amino acids in the present invention are all determined according to the EU index of KABAT numbering.
  • the mutation is I253N, that is, Ile253 is replaced with asparagine (N).
  • heterologous antibodies include, but are not limited to, bispecific antibodies, monovalent antibodies, Fc fusion proteins, and the like. That is, the heterologous antibody in the present invention may be a bispecific antibody, a monovalent antibody, or an Fc fusion protein.
  • the second aspect of the present invention is to provide a heterologous antibody.
  • the heterologous antibody contains two heavy chains with different amino acid sequences, one of which has a mutation in the I253 of the constant region of the heavy chain.
  • the mutation is I253N, that is, Ile253 is replaced with asparagine (N).
  • heterologous antibodies include, but are not limited to, bispecific antibodies, monovalent antibodies, Fc fusion proteins, and the like. That is, the heterologous antibody in the present invention may be a bispecific antibody, a monovalent antibody, or an Fc fusion protein.
  • the third aspect of the present invention is to provide a nucleotide sequence encoding the mutated heavy chain of I253 in the heterologous antibody of the second aspect of the present application.
  • the fourth aspect of the present invention is to provide a recombinant vector containing the nucleotide sequence described in the third aspect of the present invention.
  • the expression vector used in each of the above-mentioned recombinant vectors is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and / Or expression vector of sequence and other appropriate sequence.
  • the expression vector may be a virus or a plasmid, such as an appropriate phage or phagemid.
  • Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
  • the fifth aspect of the present invention is to provide a recombinant host cell containing the recombinant vector of the fourth aspect of the present invention.
  • the original host cell of the recombinant host cell of the present invention can be various conventional host cells in the art, as long as it can make the above-mentioned recombinant vector stably replicate by itself, and the nucleotides carried can be effectively expressed.
  • the original host cell may be a prokaryotic expression cell or a eukaryotic expression cell.
  • the host cell preferably includes: COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5 ⁇ , BL21 (DE3 ) Or TG1, more preferably E. coli TG1, BL21 (DE3) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies).
  • the aforementioned expression vector is transformed into a host cell to obtain the preferred recombinant host cell of the present invention.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an
  • the original host cell of the recombinant host cell is preferably a eukaryotic cell, and more preferably a CHO cell or 293E cell.
  • the sixth aspect of the present invention is to provide a composition
  • a composition comprising: (1) the heterologous antibody according to the second aspect of the present invention, and (2) a pharmaceutically acceptable carrier and/or diluent and/ Or excipients.
  • protein A affinity chromatography is a necessary purification step in the commercial preparation of IgG antibodies.
  • additional purification steps of ion exchange chromatography are often required, which increases the complexity and cost of preparation.
  • the inventors of the present invention found that only by introducing a mutation in the heavy chain of an antibody and mutating the Ile at position 253 of the EU number in the constant region of the heavy chain to Asn, the high-purity bispecificity can be obtained only through the protein A affinity layer.
  • Antibody Furthermore, this method is also applicable to other heterologous antibodies with high purity obtained only by protein A affinity chromatography.
  • the heterologous antibody refers to the antibody containing two heavy chains with different amino acid sequences, including but not limited to Bispecific antibodies, monovalent antibodies, Fc fusion proteins, etc.
  • the present invention has the following beneficial effects:
  • Figure 1 shows the binding sites of antibody Fc to protein A and FcRn.
  • the overlined marks are the amino acids on the antibody Fc that interact with protein A
  • the underlined marks are the amino acids on the antibody Fc that interact with FcRn.
  • Figure 2 is a crystal structure diagram of antibody Fc and protein A complex, underlined is I253 on antibody Fc.
  • Figure 3 is a crystal structure diagram of the complex of antibody Fc and FcRn, underlined is I253 on antibody Fc.
  • Figure 4 is a schematic diagram of heterologous antibodies and homologous antibodies.
  • Figure 5 shows the SDS-PAGE detection results of I253Q, I253K and I253R mutant antibodies. Elute directly with pH2.0 eluent, from left to right are marker, #0202, #0204, #0208, #0205, #0209, #0203, #0207, #0201.
  • Figure 6 shows the SDS-PAGE detection result of #0202.
  • (scFv) 2 -IgG wt/wt molecular weight is 198kD
  • scFv-IgG wt/I253N molecular weight is 169kD
  • IgG I253N/I253N molecular weight is 144kD
  • scFv-HC wt molecular weight is 75kD
  • HC I253N molecular weight is 50kD
  • LC molecular weight is 25kD.
  • Control is the component eluted directly with pH2.0 eluent
  • 5.5-2.0 are the components eluted with pH5.5-pH2.0 respectively.
  • Figure 7 shows the SDS-PAGE detection results of #0501 and #0502. From left to right, the lanes are #0501 directly eluted with pH3.5 fractions, #0502 directly eluted with pH3.5 fractions (control), #0502 eluted fractions with pH5-pH3.5 and marker.
  • Figure 8 shows the SDS-PAGE detection results of #0503 and #0504. From left to right, the lanes are #0503 directly eluted with pH3.5 fractions, #0504 directly eluted with pH3.5 fractions (control), #0504 eluted fractions with pH5-pH3.5 and marker.
  • Figure 9 shows the SDS-PAGE detection results of #0505 and #0506. From left to right, the lanes are #0505 directly with pH3.5 elution component, #0506 directly with pH3.5 elution component (control), #0506 with pH5-pH3.5 elution component and marker.
  • Figure 10 shows the SDS-PAGE detection results of #0601 and #0602.
  • the lanes from left to right are #0601 directly eluted with pH3.5, #0601 eluted with pH5.0-pH4.0, marker, #0602 directly eluted with pH3.5, #0602 The fraction was eluted with pH5.0-pH4.0.
  • 293E cells from NRC Biotechnology Research Institute.
  • PBS purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number B548117.
  • Citric acid purchased from Sinopharm Chemical Reagent Co., Ltd.
  • Prime star HS DNA polymerase purchased from Takara, product number R010A.
  • Endotoxin-free plasmid large-scale extraction kit purchased from TIANGEN company, item number DP117.
  • HiTrap MabSelectSuRe column purchased from GE Company.
  • AKTA-FPLC fast protein liquid chromatography system purchased from GE Company.
  • Chemidoc MP gel imager purchased from Bio-Rad.
  • G1600AX capillary electrophoresis instrument purchased from Agilent.
  • Figure 2 shows the crystal structure of the complex of Fc and protein A (PDB code: 4WWI), M252, I253, S254, L309, H310, Q311, L314, N315, K317, H433, N434, H435, Y436 on the Fc segment of the antibody (EU numbering) is located on the Fc and protein A interaction interface.
  • PDB code 4WWI
  • M252, I253, S254, L309 H310, Q311, L314, N315, K317, H433, N434, H435, Y436 on the Fc segment of the antibody (EU numbering) is located on the Fc and protein A interaction interface.
  • Figure 3 shows the crystal structure of the complex of Fc and FcRn (PDB code: 4N0U), M252, I253, S254, T256, L309, H310, Q311, L314, N315, K317, H433, N434, H435, Y436 (EU numbering) Located on the Fc and FcRn interaction interface.
  • the present invention creatively proposes that the mutation of I253 into positively charged amino acids, such as Lys and Arg, or the mutation of I253 into polar amino acids such as Asn and Gln, may make the interaction between Fc and protein A polarized- Water transfer repels, thereby changing the affinity of Fc and protein A.
  • Example 1 It can be seen from Example 1 that mutating I253 of the antibody heavy chain into positively charged amino acids, such as Lys, Arg, or polar amino acids such as Asn, Gln, may weaken the interaction between the antibody and protein A, while reducing as much as possible The effect on the binding of antibodies to FcRn. Therefore, in this example, a series of PD1 ⁇ Her2 bispecific antibody molecules with I253 mutation were constructed. As shown in Figure 4, one heavy chain of the bispecific antibody has a I253 mutation; the other heavy chain does not have a I253 mutation, and a single-chain antibody fragment is connected in series at the N-terminus of the heavy chain. In this way, the molecular weights of bispecific antibodies and two homologous antibodies can be distinguished.
  • the heterologous antibodies or heterodimers described in this embodiment refer to bispecific antibodies, and homologous antibodies or homodimers are non-target antibodies or impurities.
  • the CD3 ⁇ Her2 bispecific antibody was constructed as follows.
  • the antibody light chain variable region uses the following variable regions: TraVL (light chain variable region of anti-human Her2 antibody Trastuzumab), CD3VL (light chain variable region of anti-human CD3 antibody).
  • the antibody heavy chain variable region uses the following variable regions: TraVH (the heavy chain variable region of the anti-human Her2 antibody Trastuzumab), CD3VH (the heavy chain variable region of the anti-human CD3 antibody).
  • the CD3 antibody heavy chain variable region VH and light chain variable region VL are connected through a linker (GGGGS) 4 to obtain a single-chain variable region CD3scFv.
  • the CD3scFv and the N-terminus of the heavy chain of the anti-human Her2 antibody Trastuzumab are connected through a linker (GGGGS) 3 is connected to obtain the sequence ZY2 (SEQ ID NO:1).
  • the Trastuzumab heavy chain was introduced into the I253N point mutation (EU numbering) to obtain the antibody heavy chain ZY3, the Trastuzumab heavy chain was introduced into the I253Q point mutation (EU numbering) to obtain the antibody heavy chain ZY4, and the Trastuzumab heavy chain was introduced into the I253K point mutation (EU numbering) ,
  • the antibody heavy chain ZY5 is obtained, and the Trastuzumab heavy chain is introduced into the I253R point mutation (EU numbering) to obtain the antibody heavy chain ZY6;
  • the Trastuzumab light chain is ZY7 (SEQ ID NO: 2).
  • the gene table of each antibody is as follows:
  • Each antibody gene was inserted into an animal cell expression vector and transferred to 293 cells for expression.
  • the antibody gene was transferred to 293E cells. After 7 days of cell culture, the culture solution was subjected to high-speed centrifugation and vacuum filtration through a microporous membrane, and then loaded onto the HiTrap MabSelect SuRe column (purchased from GE). The phased use is shown in Table 3. Washing 1, eluting 1-5 to purify the antibody protein in Table 2, after eluting, neutralize the pH with a Tris buffer of pH 9.0. The eluted fractions were collected and concentrated, and reduced protein electrophoresis loading buffer and non-reduced protein electrophoresis loading buffer were added respectively, and SDS-PAGE detection was performed after boiling.
  • the eluate was eluted with pH2.0 eluent, and the eluted fractions were detected by non-reducing SDS-PAGE.
  • #0202 contained three different molecular weight antibody molecules, the molecular weights from large to small were Homologous antibody (scFv) 2 -IgG wt/wt , heterologous antibody scFv-IgG wt/I253N and homologous antibody IgG I253N/I253N .
  • #0201 is used as a reference for homologous antibody (scFv) 2 -IgG wt/wt.
  • #0202 was eluted with a gradient pH eluent, and each component was detected by SDS-PAGE. It was found that as the elution condition changed from elution 1 to elution 5, that is, with the elution The pH of the deliquation gradually decreases, and the elution components change from the homologous antibody IgG I253N/I253N to the heterologous antibody scFv-IgG wt/I253N , and then into the homologous antibody (scFv) 2 -IgG wt/wt .
  • the elution order depends on the binding strength of the eluted components and protein A, that is, the homologous antibody IgG I253N/I253N has the weakest binding capacity, so it is the first under high pH conditions (pH5.5-pH4.5) Elution; Homologous antibody (scFv) 2- IgG wt/wt has the strongest binding capacity and therefore eluted the latest under low pH conditions (pH4.0-pH2.0); heterologous antibody scFv-IgG wt/I253N binding capacity Located between the first two, it eluted under moderate pH conditions (pH4.5-pH4.0).
  • I253N point mutation does disrupt the binding of the Fc segment to protein A, so that there are 0 binding sites for IgG I253N/I253N and protein A, and 1 binding site for scFv-IgG wt/I253N and protein A, (scFv) There are 2 binding sites for 2-IgG wt/wt and protein A.
  • the more protein A binding sites the stronger the binding ability, which requires low pH eluent for elution; the fewer protein A binding sites, the weaker the binding ability, and the high pH eluent can be eluted.
  • Example 4 Increasing the association rate of the heavy chain heterodimer can further improve the purity of the heterologous antibody
  • heterologous antibodies or heterodimers described in this embodiment refer to bispecific antibodies, and homologous antibodies or homodimers are non-target antibodies or impurities.
  • Example 3 From Example 3, it is found that the components in #0202 are eluted with gradient pH eluent, and the CD3 ⁇ Her2 bispecific antibody ZY2/ZY3/ZY7 can be isolated only through the protein A purification step, but it is homologous Antibodies ZY2/ZY7 and ZY3/ZY7 still account for a large proportion of the total amount of antibodies. Theoretically, when preparing a bispecific antibody assembled from two different heavy chains and a common light chain without any modification, the bispecific antibody only accounts for about 50% of the total antibody.
  • the bispecific antibody ZY2/ZY3/ZY7 in Example 3 was mainly eluted in the eluate at pH 4.0, but a small amount of homologous antibody ZY3/ZY7 was mixed in the eluted fraction. If the association rate of the heavy chain heterodimer can be increased, the proportion of the heterologous antibody in the total antibody can be greatly increased, and the purity of the bispecific antibody can be improved at the same time.
  • the following antibody genes were constructed and used: link TraVH and IgG1 constant region to obtain antibody heavy chain ZY8; introduce Y349C, T366S, L368A, Y407V point mutations (EU numbering) in ZY8 constant region to obtain antibody heavy chain ZY9; in ZY8 constant region Introduce I253N, Y349C, T366S, L368A, Y407V point mutations (EU numbering) to obtain the antibody heavy chain ZY10; introduce S354C, T366W point mutations (EU numbering) in the ZY2 constant region to obtain the antibody heavy chain ZY11; introduce E357K to the ZY8 constant region , D399K, Y349C, T366S, L368A, Y407V point mutations (EU numbering) to obtain the antibody heavy chain ZY12; the introduction of I253N, E357K, D399K, Y349C, T366S, L368A, Y
  • Each antibody gene was inserted into an animal cell expression vector and transferred to 293 cells for expression.
  • Example 3 Wash 1 and elution 1-4 are shown for purification, and control samples #0501, #0503, and #0505 are purified by washing 1 and elution 4.
  • Example 3 the protein eluted with the pH 2.0 citric acid eluate was degraded to a certain extent, so in this example, the pH 3.5 citric acid eluate was finally used for elution.
  • Sample #0501 uses knob-into-hole technology to increase the association rate of heavy chain heterodimers. Introduce T366W point mutation (EU numbering) in the constant region of the heavy chain ZY2 to obtain a heavy chain ZY11 with a knob structure, and introduce T366S, L368A, Y407V point mutations (EU numbering) in the constant region of the heavy chain ZY8 to obtain a hole structure Heavy chain ZY9. Furthermore, the Ser with EU numbering at position 354 on the heavy chain ZY11 was mutated to Cys, and the Tyr with EU numbering at position 349 on the heavy chain ZY9 was mutated to Cys, so that the pairing between ZY11 and ZY9 was stabilized through the disulfide bond.
  • EU numbering T366W point mutation
  • L368A L368A
  • Y407V point mutations EU numbering
  • Table 7 and Figure 7 show that the use of knob-into-hole technology increases the proportion of heterologous antibody scFv-IgG (ZY9/ZY11/ZY7) to 72.85% of the total antibody.
  • the main by-product of the knob-into-hole technology comes from the hole-hole homodimer. In sample #0501, this part of the by-product accounted for 27.15% of the total antibody. If the I253N point mutation (EU numbering) is introduced into the heavy chain ZY9 with the hole structure to obtain the heavy chain ZY10, the hole-hole homodimer can be removed by the method of stepwise elution with gradient pH eluent.
  • Sample #0503 introduces electrostatic interaction on the basis of knob-into-hole technology to further improve the association rate of heavy chain heterodimer.
  • the E357K and D399K point mutations were introduced in the constant region of the heavy chain ZY9 to obtain the heavy chain ZY12 with a hole structure and positive charge
  • the K370E and K409D point mutations were introduced in the constant region of the heavy chain ZY11.
  • a heavy chain ZY14 with a knob structure and a negative charge is obtained.
  • Table 7 and Figure 8 show that the ratio of hole-hole homodimer ZY12/ZY7 decreased to 5.35%, while the ratio of heterologous antibody scFv-IgG (ZY12/ZY14/ZY7) increased to 94.55%.
  • the I253N point mutation (EU numbering) was introduced into the heavy chain ZY12 to obtain the heavy chain ZY13, and the hole-hole homodimer was further removed by the method of stepwise elution with gradient pH eluent. As shown in Figure 8, the hole-hole homodimer ZY13/ZY7 in sample #0504 was removed under the elution of pH 5.0 and pH 4.5.
  • Figure 8 and Table 7 show that the elution of pH 4.0
  • the purity of the heterologous antibody scFv-IgG (ZY13/ZY14/ZY7) in the de-fractionation was increased to 99.02%.
  • Sample #0505 introduced E357S, S364R point mutations (EU numbering) in the heavy chain ZY2 constant region to obtain a positively charged heavy chain ZY15, and introduced K370D, K409F, F405E point mutations (EU numbering) in the heavy chain ZY constant region to obtain The negatively charged heavy chain ZY17.
  • Table 7 and Figure 9 show that the purity of the heterologous antibody scFv-IgG (ZY15/ZY17/ZY7) accounted for 95.24% of the total amount of intact antibody after one step elution with a pH 3.5 citric acid eluate.
  • the main by-products of #0505 are homodimer ZY15/ZY7 and half-antibody ZY15/ZY7.
  • I253N point mutation (EU numbering) was introduced in the constant region of heavy chain ZY15 to obtain heavy chain ZY16 .
  • the homodimer ZY16/ZY7 and half-antibody ZY16/ZY7 in sample #0506 were removed under the elution of the eluate at pH 5.0 and pH 4.5.
  • Figure 9 and Table 7 show that the pH is 4.
  • the purity of the heterologous antibody scFv-IgG (ZY16/ZY17/ZY7) in the 0 elution fraction increased to 99.61%.
  • the association rate of the heavy chain heterodimer can significantly increase the heterologous antibody, that is, the double The proportion of specific antibodies.
  • the by-product is mainly one of two homologous antibodies
  • the stepwise elution method using gradient pH eluent can be efficiently and conveniently removed Homologous antibodies. This method can increase the purity of the bispecific antibody to more than 99% by only purifying protein A.
  • Example 5 Obtaining monovalent antibodies through one-step protein A purification
  • heterologous antibodies or heterodimers described in this embodiment refer to monovalent antibodies, and homologous antibodies or homodimers are non-target antibodies or impurities.
  • the two heavy chains of ordinary antibodies each contain a variable region, which binds to the antigen in a bivalent form.
  • This example studies the production of antibodies that bind to antigens in a monovalent form.
  • one of the two heavy chains is required to be a normal heavy chain, and the other is a heavy chain Fc fragment lacking the variable region and the CH1 domain. Therefore, it involves the heterologous association and purification of two different heavy chains. .
  • the required antibody genes are shown in Table 8.
  • K370D, K409F, and F405E point mutations were introduced into the Fc region of the IgG1 heavy chain (Asp at position 221 to Lys at position 447) to obtain antibody heavy chain fragment ZY18.
  • Each antibody gene was inserted into an animal cell expression vector, and then transferred to 293E cells for expression according to the combination scheme shown in Table 9.
  • the culture solution was subjected to high-speed centrifugation, vacuum filtration through a microporous membrane, and then loaded onto a HiTrap MabSelect SuRe column (purchased from GE).
  • sample #0602 use the wash 1 and wash shown in Table 6 in stages. Remove 1-4 for purification, and use wash 1 and elution 4 for sample #0601. After elution, the pH was neutralized with a Tris buffer of pH 9.0. The eluted fractions were collected and concentrated, and non-reducing protein electrophoresis loading buffer was added respectively, and SDS-PAGE detection was performed after boiling. The eluted components were analyzed by non-reducing capillary gel electrophoresis, and the percentage of peak area of each product component was calculated.
  • the main by-products of sample #0601 were homodimer IgG (ZY15/ZY7) and half-antibody (ZY15/ZY7), and no by-product Fc (ZY18/ZY7) was observed. Since homodimer IgG (ZY15/ZY7) and heterologous antibody ZY15/ZY18/ZY7 have the same number of protein A binding sites, the stepwise elution method using gradient pH eluent cannot effectively remove homology. Dimer IgG (ZY15/ZY7) and half antibody (ZY15/ZY7). The I253N point mutation (EU numbering) was introduced into the constant region of the heavy chain ZY15 to obtain the heavy chain ZY16.
  • EU numbering was introduced into the constant region of the heavy chain ZY15 to obtain the heavy chain ZY16.
  • the homodimer (ZY16/ZY7) and half-antibody (ZY16/ZY7) were removed by the method of stepwise elution with gradient pH eluent. ). As shown in Figure 10, the homodimer ZY16/ZY7 and half-antibody ZY16/ZY7 in sample #0602 were removed under the elution of pH 5.0 and pH 4.5. Figure 10 and Table 10 show that the pH is 4. The purity of the heterologous antibody Fab-Fc (ZY16/ZY18/ZY7) in the 0 elution fraction increased to 99.53%.
  • monovalent antibodies are not limited to one form of Fab-Fc introduced in this embodiment, and other forms of monovalent antibodies such as scFv-Fc fusion proteins are also suitable for preparation using the method of the present invention. More broadly, any antibody or Fc fusion protein containing only CH2 and CH3 domains in one of the heavy chains can be prepared by the method of the present invention.

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Abstract

一种得到高纯度异源抗体的方法，所述异源抗体含有两条氨基酸序列不同的重链，在其中一条重链恒定区的I253引入突变，进而通过蛋白A亲和层析纯化，即可获得高纯度的双特异性抗体，其中，以上所述的氨基酸的位置根据KABAT编号的EU索引确定。所述方法仅在重链引入一个突变，将重链恒定区中的EU编号第253位的Ile突变为Asn，即可只通过一步蛋白A亲和层就能将异源抗体的纯度提高到99％以上，简化了纯化步骤，降低制备成本。

Description

一种得到高纯度异源抗体的方法 技术领域
本发明属于抗体工程领域，具体涉及一种得到高纯度异源抗体的方法，所述异源抗体含有两条氨基酸序列不同的重链。
背景技术
双特异性抗体有多种构建方式，其中IgG型双特异性抗体具有和普通抗体相似的结构、理化性质和Fc段功能。通常IgG型双特异性抗体由两条氨基酸序列不同的重链(即抗抗原A的重链和抗抗原B的重链)和两条氨基酸序列不同的轻链(即抗抗原A的轻链和抗抗原B的轻链)组成。当4条多肽链组合时，会产生8种不同的组合方式，其中只有一种为所需要的目标抗体分子。而从8种分子中分离纯化得到目标分子效率极低且非常困难。
作为解决该问题的方法，文献1报道了使用共同重链和两条不同轻链组合的方法。但该方法的局限性在于，要筛选得到氨基酸序列完全相同的抗抗原A的重链和抗抗原B的重链非常困难。文献2、3报道了使用共同轻链和两条不同重链组合的方法。但该方法的局限性在于，2条不同的重链会有3种组合方式，包括一种异源抗体(双特异性抗体)和两种同源抗体(杂质)，其中目标双特异性抗体的比例理论上通常只有约50％。如果提高2条重链异源缔合的效率，则可将目标双特异性抗体的表达效率提高到90-95％(文献4)。为了进一步去除两种同源抗体，文献5报道了下述方法：通过在两种重链的可变区引入氨基酸突变赋予两种同源抗体和目标双特异性抗体不同的等电点，从而通过离子交换层析纯化得到目标双特异性抗体。然而需要注意的是，在IgG型抗体商业化制备过程中，离子交换层析并非必需的纯化步骤，增加离子交换层析固然可以提高目标抗体的纯度，但也降低了最终的产率，导致制备成本增加。
蛋白A亲和层析作为IgG型抗体必需的纯化步骤，如何通过蛋白A亲和层析得到高纯度的双特异性抗体成为研究的新方向。文献6报道了一种方法：使用由小鼠IgG2a的重链(可与蛋白A结合)和大鼠IgG2b的重链(不与蛋白A结合)组成双特异性抗体，仅利用蛋白A纯化即可将目标双特异性抗体纯化至95％的纯度。但该方法的局限性在于，小鼠和大鼠的重链恒定区免疫原性极高(文献7)，而且利用该方法制备的抗体卡妥索单抗(Catumaxomab)在人体内半衰期为约2.1天，与通常的人IgG半衰期2-3周相比极短(文献8)。文献9报道 了另一种利用蛋白A纯化得到高纯度的双特异性抗体的方法：在构成双特异性抗体的其中一条重链恒定区上引入突变。具体来说，将EU编号第435位的His突变为Arg，从而改变与蛋白A的结合力，能将目标抗体纯化至93-99.6％不等的纯度。
文献1：Fischer,N.,et al.(2015).Nat Commun 6:6113.
文献2：WO98050431
文献3：WO2006109592
文献4：WO2006106905
文献5：WO2007114325
文献6：WO95033844
文献7：Clin Cancer Res 2007 13:3899-3905
文献8：J Clin Oncol 26:2008(May 20suppl；abstr 14006)
文献9：WO2011078332
发明内容
本发明的目的在于克服现有技术中的不足，提供一种仅利用蛋白A亲和层析即可得到高纯度异源抗体的方法。本发明通过在重链恒定区引入新的突变，改变与蛋白A的结合力，进而通过蛋白A亲和层析，即可获得纯度>99％的双特异性抗体。
本发明的第一个方面是提供一种得到高纯度异源抗体的方法，所述异源抗体含有两条氨基酸序列不同的重链，在其中一条重链恒定区的I253引入突变，进而通过蛋白A亲和层析纯化，即可获得高纯度的双特异性抗体，其中，以上所述的氨基酸的位置根据KABAT编号的EU索引确定。
本发明中的氨基酸的位置均根据KABAT编号的EU索引确定。
优选地，所述突变为I253N，即Ile253被替换为天冬酰胺(N)。
其中，所述异源抗体包括但不限于为双特异性抗体、一价抗体、Fc融合蛋白等。即本发明中所述异源抗体可以为双特异性抗体、一价抗体、或Fc融合蛋白等。
本发明的第二个方面是提供一种异源抗体所述异源抗体含有两条氨基酸序列不同的重链，其中一条重链恒定区的I253发生突变。
优选地，所述突变为I253N，即Ile253被替换为天冬酰胺(N)。
其中，所述异源抗体包括但不限于为双特异性抗体、一价抗体、Fc融合蛋白等。即本发明中所述异源抗体可以为双特异性抗体、一价抗体、或Fc融合蛋白等。
本发明的第三个方面是提供一种编码本申请第二个方面所述异源抗体中I253发生突变的重链的核苷酸序列。
本发明的第四个方面是提供含有本发明第三个方面所述的核苷酸序列的重组载体。
其中，上述各重组载体所使用的表达载体为本领域常规的表达载体，是指包含适当的调控序列，例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒，如适当的噬菌体或者噬菌粒，更多技术细节请参见例如Sambrook等，Molecular Cloning：A Laboratory Manual，第二版，Cold Spring Harbor Laboratory Press，1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology，第二版，Ausubel等编著。本发明对此不作特别限定。
本发明的第五个方面是提供含有本发明第四个方面所述重组载体的重组宿主细胞。
本发明所述的重组宿主细胞的原始宿主细胞可以为本领域常规的各种宿主细胞，只要能满足使上述重组载体稳定地自行复制，且所携带所述的核苷酸可被有效表达即可。其中所述原始宿主细胞可以原核表达细胞或真核表达细胞，所述宿主细胞较佳地包括：COS、CHO(中国仓鼠卵巢，Chinese H amster Ovary)、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1，更佳地为E.coli TG1、BL21(DE3)细胞(表达单链抗体或Fab抗体)或者CHO-K1细胞(表达全长IgG抗体)。将前述表达载体转化至宿主细胞中，即可得本发明优选的重组宿主细胞。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
作为优选的方案，所述重组宿主细胞的原始宿主细胞优选为真核细胞，进一步优选为CHO细胞或293E细胞。
本发明的第六个方面是提供一种组合物，其含有：(1)本发明第二个方面所述的异源抗体，以及(2)药学上可接受的载体和/或稀释剂和/或赋形剂。
通常，蛋白A亲和层析是IgG型抗体商业化制备过程中必需的纯化步骤。然而，为了制备高纯度的双特异性抗体，往往需要追加离子交换层析的纯化步骤，这增加了制备的复杂性和成本。本发明的发明人发现，仅在抗体的重链引入一个突变，将重链恒定区中的EU编号第253位的Ile突变为Asn，即可仅通过蛋白A亲和层获得高纯度双特异性抗体。更进一步的，该方法同样适用于仅通过蛋白A亲和层析纯化得到高纯度的其它异源抗体，所指的异源抗体指抗体含有两条氨基酸序列不同的重链，包括而不局限于双特异性抗体、一价抗体、Fc融合蛋白等。
与现有技术相比，本发明的有益效果：
本发明仅在重链引入一个突变，将重链恒定区中的EU编号第253位的Ile突变为Asn，即可只通过一步蛋白A亲和层就能将异源抗体的纯度提高到99％以上，大大简化了纯化步骤，降低制备成本，具有广阔的商业应用前景。
附图说明
图1为抗体Fc与蛋白A和FcRn的结合位点。上划线标记的是抗体Fc上与蛋白A相互作用的氨基酸，下划线标记的是抗体Fc上与FcRn相互作用的氨基酸。
图2为抗体Fc与蛋白A复合物晶体结构图，下划线标记的是抗体Fc上的I253。
图3为抗体Fc与FcRn复合物晶体结构图，下划线标记的是抗体Fc上的I253。
图4为异源抗体和同源抗体示意图。
图5为I253Q、I253K和I253R突变抗体的SDS-PAGE检测结果。直接用pH2.0的洗脱液洗脱，从左至右分别为marker、#0202、#0204、#0208、#0205、#0209、#0203、#0207、#0201。
图6为#0202的SDS-PAGE检测结果。(scFv) 2-IgG wt/wt分子量为198kD，scFv-IgG wt/I253N分子量为169kD，IgG I253N/I253N分子量为144kD，scFv-HC wt分子量为75kD，HC I253N分子量为50kD，LC分子量为25kD。Control为直接用pH2.0的洗脱液洗脱的组分，5.5-2.0分别为pH5.5-pH2.0洗脱的组分。
图7为#0501、#0502的SDS-PAGE检测结果。泳道从左至右分别为#0501直接用pH3.5洗脱组分、#0502直接用pH3.5洗脱组分(对照)、#0502用pH5-pH3.5洗脱组分和marker。
图8为#0503、#0504的SDS-PAGE检测结果。泳道从左至右分别为#0503直接用pH3.5洗脱组分、#0504直接用pH3.5洗脱组分(对照)、#0504用pH5-pH3.5洗脱组分和marker。
图9为#0505、#0506的SDS-PAGE检测结果。泳道从左至右分别为#0505直接用pH3.5洗脱组分、#0506直接用pH3.5洗脱组分(对照)、#0506用pH5-pH3.5洗脱组分和marker。
图10为#0601、#0602的SDS-PAGE检测结果。泳道从左至右分别为#0601直接用pH3.5洗脱组分、#0601用pH5.0-pH4.0洗脱组分、marker、#0602直接用pH3.5洗脱组分、#0602用pH5.0-pH4.0洗脱组分。
具体实施方式
以下实施例、实验例是对本发明进行进一步的说明，不应理解为是对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因 插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:A Laboratory Manual,2 nd edition,Cold spring Harbor Laboratory Press.
以下实施例中使用的实验材料和来源以及实验试剂的配制方法具体说明如下。
1、实验材料：
293E细胞：来自NRC biotechnology Research Institute。
2、实验试剂：
PBS：购自生工生物工程(上海)股份有限公司，货号B548117。
柠檬酸：购自国药集团化学试剂有限公司。
Prime star HS DNA polymerase：购自Takara公司，货号R010A。
无内毒素质粒大提试剂盒：购自TIANGEN公司，货号DP117。
3、实验仪器：
HiTrap MabSelectSuRe柱：购自GE公司。
AKTA-FPLC快速蛋白液相色谱系统：购自GE公司。
C1000Touch Thermal Cycler PCR仪：购自Bio-Rad公司。
Chemidoc MP凝胶成像仪：购自Bio-Rad公司。
离心机：购自Eppendorf公司。
G1600AX毛细管电泳仪：购自安捷伦公司。
实施例1.重链恒定区氨基酸修饰的设计
抗体Fc段与蛋白A和FcRn结合的位点如图1所示。图2显示了Fc与蛋白A的复合物晶体结构(PDB code:4WWI)，抗体Fc段上的M252、I253、S254、L309、H310、Q311、L314、N315、K317、H433、N434、H435、Y436(EU编号)位于Fc和蛋白A相互作用界面上。图3显示了Fc与FcRn的复合物晶体结构(PDB code:4N0U)，M252、I253、S254、T256、L309、H310、Q311、L314、N315、K317、H433、N434、H435、Y436(EU编号)位于Fc和FcRn相互作用界面上。本发明创造性的提出，将I253突变成带正电荷的氨基酸，如Lys、Arg，或者将I253突变成极性氨基酸如Asn、Gln，可能会使Fc与蛋白A的相互作用产生极性-输水排斥，从而改变Fc与蛋白A的亲和力。
实施例2.抗体表达载体的构建和表达
由实施例1可知，将抗体重链的I253突变成带正电荷的氨基酸，如Lys、Arg，或极性氨基酸如Asn、Gln，有可能减弱抗体与蛋白A的相互作用，同时尽可能减少对抗体与FcRn结合的影响。因此，本实施例构建一系列带有I253突变的PD1×Her2双特异性抗体分子。如图4所示，该双特异性抗体的一条重链带有I253突变；另一条重链不带I253突变，并且在重链的N端串联单链抗体片段。这样使得双特异性抗体和2种同源抗体的分子量得到区分。本实施例中所描述的异源抗体或异源二聚体即指的是双特异性抗体，同源抗体或同源二聚体则为非目的抗体或杂质。
CD3×Her2双特异性抗体构建如下。抗体轻链可变区使用下述可变区：TraVL(抗人Her2抗体Trastuzumab的轻链可变区)，CD3VL(抗人CD3抗体的轻链可变区)。抗体重链可变区使用下述可变区：TraVH(抗人Her2抗体Trastuzumab的重链可变区)，CD3VH(抗人CD3抗体的重链可变区)。
将CD3抗体重链可变区VH和轻链可变区VL通过连接接头(GGGGS) 4连接起来，得到单链可变区CD3scFv，将CD3scFv和抗人Her2抗体Trastuzumab的重链N端通过连接接头(GGGGS) 3连接起来，得到序列ZY2(SEQ ID NO：1)。
将Trastuzumab重链引入I253N点突变(EU编号)，得到抗体重链ZY3，将Trastuzumab重链引入I253Q点突变(EU编号)，得到抗体重链ZY4，将Trastuzumab重链引入I253K点突变(EU编号)，得到抗体重链ZY5，将Trastuzumab重链引入I253R点突变(EU编号)，得到抗体重链ZY6；Trastuzumab轻链为ZY7(SEQ ID NO：2)。各抗体基因表记如下：
表1：
编号 分子形式
CD3scFv CD3VH-(GGGGS) 4-CD3VL
ZY1 CD3scFv-(GGGGS) 3-TraVH
ZY2 scFv-HC wt
ZY3 HC I253N
ZY4 HC I253Q
ZY5 HC I253K
ZY6 HC I253R
ZY7 TraVL-Cκ
将各抗体基因插入动物细胞表达载体中，顺转到293细胞中表达。各抗体基因的组合如下表2所示，转染比例为重链1：重链2：轻链＝1:1:1。
表2：
Figure PCTCN2020140756-appb-000001
实施例3.蛋白A亲和层析洗脱条件的研究
抗体基因顺转到293E细胞中，细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后上样至HiTrap MabSelectSuRe柱(购自GE公司)，阶段性的使用表3所示的清洗1、洗脱1-5对表2中的抗体蛋白进行纯化，洗脱后用pH9.0的Tris缓冲液中和pH。收集洗脱组分并浓缩，分别加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液，煮沸后进行SDS-PAGE检测。
表3
平衡化 PBS
清洗1 PBS
洗脱1 100mM柠檬酸，pH5.5
洗脱2 100mM柠檬酸，pH5.0
洗脱3 100mM柠檬酸，pH4.5
洗脱4 100mM柠檬酸，pH4.0
洗脱5 100mM柠檬酸，pH2.0
如图5所示，用pH2.0的洗脱液洗脱，对洗脱组分进行非还原SDS-PAGE检测，发现#0202包含了三种不同分子量的抗体分子，分子量从大到小分别为同源抗体(scFv) 2-IgG wt/wt、异源抗体scFv-IgG wt/I253N和同源抗体IgG I253N/I253N。#0201作为同源抗体(scFv) 2-IgG wt/wt的对照品。然而，#0203、#0204、#0205只得到了同源抗体(scFv) 2-IgG wt/wt，并且有重链二聚体、未配对的重链和轻链等抗体片段出现，提示ZY4、ZY5、ZY6中引入的I253Q、I253K和I253R点突变可能影响了轻重链的组装。进一步纯化#0207、#0208、#0209发现，确实只得到了重链二聚体和未配对的重链，表明I253Q、I253K和I253R的确影响了轻重链的组装。
如图6所示，对#0202用梯度pH的洗脱液洗脱，并对各组分进行SDS-PAGE检测，发现随着洗脱条件从洗脱1变成洗脱5，即随着洗脱液的pH逐渐降低，洗脱成分由同源抗体IgG I253N/I253N变成异源抗体scFv-IgG wt/I253N，再变成同源抗体(scFv) 2-IgG wt/wt。更具体的说，洗脱顺序取决于洗脱成分与蛋白A的结合强弱，即同源抗体IgG I253N/I253N结合力最弱因此在高pH条件下(pH5.5-pH4.5)最先洗脱；同源抗体(scFv) 2-IgG wt/wt结合力最强因此在低pH条件下(pH4.0-pH2.0)最晚洗脱；异源抗体scFv-IgG wt/I253N结合力位于前两者之间，因此在中等pH条件下(pH4.5-pH4.0)洗脱。这表明I253N点突变的确破坏了Fc段与蛋白A的结合，使得IgG I253N/I253N与蛋白A结合位点为0个，scFv-IgG wt/I253N与蛋白A结合位点为1个，(scFv) 2-IgG wt/wt与蛋白A结合位点为2个。蛋白A结合位点越多则结合能力越强，需要低pH的洗脱液洗脱；蛋白A结合位点越少则结合能力越弱，高pH的洗脱液就可以洗脱。
实施例4.提高重链异源二聚体的缔合率能进一步提高异源抗体的纯度
本实施例中所描述的异源抗体或异源二聚体即指的是双特异性抗体，同源抗体或同源二聚体则为非目的抗体或杂质。
通过实施例3发现，对#0202中各组分利用梯度pH洗脱液洗脱，可以仅通过蛋白A纯化步骤，就能分离得到CD3×Her2双特异性抗体ZY2/ZY3/ZY7，但同源抗体ZY2/ZY7和ZY3/ZY7仍占据了抗体总量的很大比重。理论上，在没有任何修饰的情况下制备由两条不同的重链和一条共同的轻链组装成的双特异性抗体时，双特异性抗体只占抗体总量的约50％。另外，实施例3中双特异性抗体ZY2/ZY3/ZY7主要在pH4.0的洗脱液中洗脱，但洗脱组分中会夹杂少量同源抗体ZY3/ZY7。如果能提高重链异源二聚体的缔合率，则能大幅度提高异源抗体在抗体总量中的比例，同时提高双特异性抗体的纯度。
构建并使用以下抗体基因：将TraVH和IgG1恒定区连接，得到抗体重链ZY8；在ZY8恒定区引入Y349C、T366S、L368A、Y407V点突变(EU编号)，得到抗体重链ZY9；在 ZY8恒定区引入I253N、Y349C、T366S、L368A、Y407V点突变(EU编号)，得到抗体重链ZY10；在ZY2恒定区引入S354C、T366W点突变(EU编号)，得到抗体重链ZY11；在ZY8恒定区引入E357K、D399K、Y349C、T366S、L368A、Y407V点突变(EU编号)，得到抗体重链ZY12；在ZY8恒定区引入I253N、E357K、D399K、Y349C、T366S、L368A、Y407V点突变(EU编号)，得到抗体重链ZY13；在ZY2恒定区引入K370E、K409D、S354C、T366W点突变(EU编号)，得到抗体重链ZY14；在ZY8恒定区引入E357S、S364R点突变(EU编号)，得到抗体重链ZY15；在ZY8恒定区引入I253N、E357S、S364R点突变(EU编号)，得到抗体重链ZY16；在ZY2恒定区引入K370D、K409F、F405E点突变(EU编号)，得到抗体重链ZY17。本实施例所需抗体基因见表4。
表4
编号 分子形式
ZY7 TraVL-Cκ
ZY8 HC wt
ZY9 HC Y349C,T366S,L368A,Y407V
ZY10 HC I253N,Y349C,T366S,L368A,Y407V
ZY11 scFv-HC S354C,T366W
ZY12 HC E357K,D399K,Y349C,T366S,L368A,Y407V
ZY13 HC I253N,E357K,D399K,Y349C,T366S,L368A,Y407V
ZY14 scFv-HC K370E,K409D,S354C,T366W
ZY15 HC E357S,S364R
ZY16 HC I253N,E357S,S364R
ZY17 scFv-HC K370D,K409F,F405E
将各抗体基因插入动物细胞表达载体中，顺转到293细胞中表达。各抗体基因的组合如下表5所示，转染比例为重链1：重链2：轻链＝1:1:1。
表5
Figure PCTCN2020140756-appb-000002
Figure PCTCN2020140756-appb-000003
细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后上样至HiTrap MabSelectSuRe柱(购自GE公司)，对测试样品#0502、#0504、#0506阶段性的使用表6所示的清洗1、洗脱1-4进行纯化，对照样品#0501、#0503、#0505使用清洗1、洗脱4进行纯化。在实施例3中，用pH2.0的柠檬酸洗脱液洗脱的蛋白存在一定的降解，因此在本实施例中最后采用pH3.5的柠檬酸洗脱液洗脱。洗脱后用pH9.0的Tris缓冲液中和pH。收集洗脱组分并浓缩，分别加入非还原型蛋白电泳上样缓冲液，煮沸后进行SDS-PAGE检测。洗脱组分用非还原毛细管凝胶电泳分析，并计算各产物组分峰面积的百分比例。
表6
平衡化 PBS
清洗1 PBS
洗脱1 100mM柠檬酸，pH5.0
洗脱2 100mM柠檬酸，pH4.5
洗脱3 100mM柠檬酸，pH4.0
洗脱4 100mM柠檬酸，pH3.5
样品#0501采用了knob-into-hole技术来提高重链异源二聚体的缔合率。在重链ZY2恒定区引入T366W点突变(EU编号)，获得带有knob结构的重链ZY11，在重链ZY8恒定区引入T366S、L368A、Y407V点突变(EU编号)，获得带有hole结构的重链ZY9。更进一步，将重链ZY11上EU编号为354位的Ser突变为Cys，将重链ZY9上EU编号为349位的Tyr突变为Cys，使得ZY11和ZY9之间的配对通过二硫键得到稳固。表7和图7显示，利用knob-into-hole技术使得异源抗体scFv-IgG(ZY9/ZY11/ZY7)占抗体总量比例提高到72.85％。knob-into-hole技术主要的副产物来自于hole-hole同源二聚体，在样品#0501中，该部分副产物占到抗体总量的27.15％。如果在带有hole结构的重链ZY9上引入I253N点突变(EU编号)获得重链ZY10，则可以利用梯度pH洗脱液阶段性洗脱的方法去除hole-hole同源二聚体。如图7显示，样品#0502中hole-hole同源二聚体ZY11/ZY7在pH5.0和pH4.5的洗脱液洗脱下被去除，图7和表7显示，pH4.0的洗脱组分中异源抗体scFv-IgG(ZY10/ZY11/ZY7)纯度提高到99.63％。
样品#0503在knob-into-hole技术的基础上引入静电相互作用来进一步提高重链异源二 聚体的缔合率。具体来说，在重链ZY9恒定区引入E357K、D399K点突变(EU编号)，获得带有hole结构和正电荷的重链ZY12，在重链ZY11恒定区引入K370E、K409D点突变(EU编号)，获得带有knob结构和负电荷的重链ZY14。表7和图8显示，hole-hole同源二聚体ZY12/ZY7比例下降到5.35％，而异源抗体scFv-IgG(ZY12/ZY14/ZY7)比例提高到94.55％。在重链ZY12上引入I253N点突变(EU编号)获得重链ZY13，利用梯度pH洗脱液阶段性洗脱的方法进一步去除hole-hole同源二聚体。如图8显示，样品#0504中hole-hole同源二聚体ZY13/ZY7在pH5.0和pH4.5的洗脱液洗脱下被去除，图8和表7显示，pH4.0的洗脱组分中异源抗体scFv-IgG(ZY13/ZY14/ZY7)纯度提高到99.02％。
样品#0505在重链ZY2恒定区引入E357S、S364R点突变(EU编号)，获得带有正电荷的重链ZY15，在重链ZY恒定区引入K370D、K409F、F405E点突变(EU编号)，获得带有负电荷的重链ZY17。表7和图9显示，经pH3.5的柠檬酸洗脱液一步洗脱，异源抗体scFv-IgG(ZY15/ZY17/ZY7)的纯度即占到完整抗体总量的95.24％。#0505的主要副产物为同源二聚体ZY15/ZY7和半抗体ZY15/ZY7，为了进一步去除这两种副产物，在重链ZY15恒定区引入I253N点突变(EU编号)，获得重链ZY16，利用梯度pH洗脱液阶段性洗脱的方法进一步去除同源二聚体ZY16/ZY7和半抗体ZY16/ZY7。如图9显示，样品#0506中同源二聚体ZY16/ZY7和半抗体ZY16/ZY7在pH5.0和pH4.5的洗脱液洗脱下被去除，图9和表7显示，pH4.0的洗脱组分中异源抗体scFv-IgG(ZY16/ZY17/ZY7)纯度提高到99.61％。
综上所述，在制备由两条不同的重链和一条共同的轻链组装成的双特异性抗体时，提高重链异源二聚体的缔合率能显著提高异源抗体，即双特异性抗体的比例。当副产物主要为两种同源抗体中的一种时，通过在该同源抗体恒定区引入I253N点突变(EU编号)，利用梯度pH洗脱液阶段性洗脱的方法可以高效方便的去除同源抗体。该方法仅通过蛋白A纯化即可提高双特异性抗体的纯度至99％以上。
表7
Figure PCTCN2020140756-appb-000004
实施例5.通过一步蛋白A纯化获得一价抗体
本实施例中所描述的异源抗体或异源二聚体即指的是一价抗体，同源抗体或同源二聚体则为非目的抗体或杂质。
普通抗体的2条重链各包含一个可变区，以二价形式与抗原结合。本实施例研究以一价形式与抗原结合的抗体制作。为了制备一价抗体，要求2条重链其中的一条是普通重链，另一条是缺失了可变区和CH1结构域的重链Fc片段，因此涉及两条不同重链异源缔合以及纯化。
所需抗体基因见表8。采用与实施例4相同的方法，向IgG1重链Fc区(EU编号第221位的Asp到447位的Lys)内引入K370D、K409F、F405E点突变(EU编号)，得到抗体重链片段ZY18。
表8
编号 分子形式
ZY7 TraVL-Cκ
ZY15 HC E357S,S364R
ZY16 HC I253N,E357S,S364R
ZY18 Fc K370D,K409F,F405E
将各抗体基因插入动物细胞表达载体中，按表9所示的组合方案顺转到293E细胞中表达。为了尽量减少副产物ZY18/ZY7，转染比例为重链1：重链2：轻链＝1.5:1:1。
表9
Figure PCTCN2020140756-appb-000005
细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后上样至HiTrap MabSelectSuRe柱(购自GE公司)，对样品#0602阶段性的使用表6所示的清洗1、洗脱1-4进行纯化，对样品#0601使用清洗1、洗脱4进行纯化。洗脱后用pH9.0的Tris缓冲液中和pH。收集洗脱组分并浓缩，分别加入非还原型蛋白电泳上样缓冲液，煮沸后进行SDS-PAGE检测。洗脱组分用非还原毛细管凝胶电泳分析，并计算各产物组分峰面积的百分比例。
如图10所示，样品#0601的主要副产物为同源二聚体IgG(ZY15/ZY7)和半抗体(ZY15/ZY7)，未观察到副产物Fc(ZY18/ZY7)。由于同源二聚体IgG(ZY15/ZY7)和异源抗体ZY15/ZY18/ZY7有相同的蛋白A结合位点个数，因此利用梯度pH洗脱液阶段性洗脱的方法不能有效去除同源二聚体IgG(ZY15/ZY7)和半抗体(ZY15/ZY7)。在重链ZY15恒定区引入I253N点突变(EU编号)，获得重链ZY16，利用梯度pH洗脱液阶段性洗脱的方法去除同源二聚体(ZY16/ZY7)和半抗体(ZY16/ZY7)。如图10显示，样品#0602中同源二聚体ZY16/ZY7和半抗体ZY16/ZY7在pH5.0和pH4.5的洗脱液洗脱下被去除，图10和表10显示，pH4.0的洗脱组分中异源抗体Fab-Fc(ZY16/ZY18/ZY7)纯度提高到99.53％。
表10
Figure PCTCN2020140756-appb-000006
本领域技术人员应该理解，一价抗体不局限于本实施介绍的Fab-Fc一种形式，其它形式的一价抗体例如scFv-Fc融合蛋白也适用于利用本发明方法进行制备。更广泛的说，凡是其中一条重链为仅包含CH2和CH3结构域的抗体或Fc融合蛋白，均可利用本发明方法进行制备。
以上对本发明的具体实施例进行了详细描述，但其只是作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。
Figure PCTCN2020140756-appb-000007
Figure PCTCN2020140756-appb-000008
Figure PCTCN2020140756-appb-000009
Figure PCTCN2020140756-appb-000010
Figure PCTCN2020140756-appb-000011
Figure PCTCN2020140756-appb-000012
Figure PCTCN2020140756-appb-000013
Figure PCTCN2020140756-appb-000014
Figure PCTCN2020140756-appb-000015

Claims (10)

  1. 一种得到高纯度异源抗体的方法，其特征在于，所述异源抗体含有两条氨基酸序列不同的重链，在其中一条重链恒定区的I253引入突变，进而通过蛋白A亲和层析纯化，即可获得高纯度的双特异性抗体，其中，以上所述的氨基酸的位置根据KABAT编号的EU索引确定。
  2. 根据权利要求1所述的方法，其特征在于，所述突变为I253N。
  3. 根据权利要求1所述的方法，其特征在于，所述异源抗体为双特异性抗体、一价抗体或Fc融合蛋白。
  4. 一种异源抗体，其特征在于，所述异源抗体含有两条氨基酸序列不同的重链，其中一条重链恒定区的I253发生突变。
  5. 根据权利要求3所述的异源抗体，其特征在于，所述突变为I253N。
  6. 根据权利要求3所述的异源抗体，其特征在于，所述异源抗体为双特异性抗体、一价抗体或Fc融合蛋白。
  7. 编码权利要求4-6中任意一项所述异源抗体中I253发生突变的重链的核苷酸序列。
  8. 含有权利要求7所述的核苷酸序列的重组载体。
  9. 含有权利要求8所述重组载体的重组宿主细胞。
  10. 一种组合物，其特征在于，其含有：(1)权利要求4-6中任意一项所述的异源抗体，以及(2)药学上可接受的载体和/或稀释剂和/或赋形剂。
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